Non-canonical Regulation of EGFR by the Air Pollutant 9,10-Phenanthrenequinone

Abstract

9,10-Phenanthrenequinone (9,10-PQ), a polycyclic aromatic hydrocarbon that is present in air pollutants, such as diesel exhaust gas and PM2.5, causes the production of excess reactive oxygen species. 9,10-PQ was recently shown to induce the activation of epidermal growth factor receptor (EGFR) by inhibiting protein tyrosine phosphatase 1B. In the present study, we focused on the non-canonical regulation of EGFR, including negative feedback and internalization. In contrast to previous findings, 9,10-PQ inhibited the constitutive tyrosine phosphorylation of EGFR via the mitogen-activated protein extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK)-mediated phosphorylation of Thr-669 in EGFR-overexpressing A431 and MDA-MB-468 cells. In addition, 9,10-PQ induced the clathrin-mediated endocytosis of EGFR via the p38 phosphorylation of Ser-1015 in HeLa and A549 cells. These results revealed that 9,10-PQ strongly induced the non-canonical regulation of EGFR by activating mitogen-activated protein kinase (MAPK).

INTRODUCTION

9,10-Phenanthrenequinone (9,10-PQ) is a polycyclic aromatic hydrocarbon that is present in air pollutants, such as diesel particular matter (DPM) and PM2.5.^1,2) It functions as an electron acceptor in cells, which produces excess reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), and induces protein oxidation and apoptosis.^3–5) Long-term exposure to DPM is also considered to induce lung cell carcinogenesis through the production of ROS.⁶⁾

Epidermal growth factor receptor (EGFR), a member of the receptor tyrosine (Tyr) kinase family, has been the focus of molecular-targeted therapy for cancer because its overexpression and mutations are involved in carcinogenesis and the progression of several types of cancer, including the adenocarcinomas of non-small cell lung cancer (NSCLC).^7–10) Ligand binding to the EGFR extracellular domain triggers the canonical activation of the intracellular tyrosine kinase domain through its asymmetric dimerization. There is increasing evidence to support the non-canonical regulation of EGFR by the serine/threonine phosphorylation of the intracellular domain.^11–25) The two most characterized phenomena are the negative feedback regulation of tyrosine kinase and clathrin-mediated endocytosis (CME), which are regulated by the extracellular signal-regulated kinase (ERK)-mediated phosphorylation of Thr-669 in the juxtamembrane domain^14,20) and the p38-mediated phosphorylation of a serine/threonine cluster, including Ser-1015, around the C-terminal clathrin-recognition site (Leu-1010/1011; LL),^11,15,21) respectively. We and others have shown that phorbol esters, inflammatory cytokines, and other cellular stresses induce these non-canonical mechanisms.^23–25)

9,10-PQ is reduced by intracellular electron donors in cells, which interact with oxygen to produce H₂O₂ and eventually inhibit protein tyrosine phosphatase 1B (PTP1B) by oxidizing the thiol group. 9,10-PQ has been shown to increase the canonical tyrosine phosphorylation of EGFR because it is one of the major PTP1B substrates.²⁶⁾ In contrast, the effects of 9,10-PQ on the non-canonical regulation of EGFR remain unclear. Therefore, we herein investigated the effects of 9,10-PQ on the two typical non-canonical mechanisms of EGFR.

MATERIALS AND METHODS

Antibodies and Reagents

Phospho-specific antibodies against p38 (Thr-180/Tyr-182), ERK (Thr-202/Tyr-204), and EGFR (Tyr-1068 and Thr-669) were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). A phospho-EGFR (Ser-1015) rabbit recombinant antibody was generated as previously reported.¹⁵⁾ Anti-EGFR (1005 and A-10) and α-tubulin (B-7) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). A monoclonal anti-EGFR antibody (clone LA1) was obtained from Merck KGaA (Darmstadt, Germany). Recombinant human EGF and tumor necrosis factor (TNF)-α were obtained from R&D Systems (Minneapolis, MN, U.S.A.). 9,10-PQ (Sigma-Aldrich; St. Louis, MO, U.S.A.), gefitinib (FUJIFILM Wako Pure Chemical Corporation; Osaka, Japan), SB203580 (BLD Pharmatech; Cincinnati, OH, U.S.A.), and trametinib (AdooQ BioScience; Irvine, CA, U.S.A.) were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was less than 0.1%.

Cell Culture

A431, MDA-MB-468, and HeLa cells were obtained from the American Type Culture Collection (ATCC, Rockville, TX, U.S.A.) and maintained in Dulbecco’s modified Eagle’s medium (high-glucose; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal calf serum, 4 mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan) at 37 °C in 5% CO₂. A549 (ATCC) cells were maintained in RPMI-1640 (Nissui Pharmaceutical) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in 5% CO₂. EGFR-knockout (EGFR-KO) HeLa cells were established by using the clustered regularly interspaced short palindromic repeats CRISPR-associated proteins 9 (CRISPR/Cas9) system and were rescued with pEGFP-N1 plasmid DNAs encoding EGFR (wild type, R1m; S1015A/T1017A/S1018A, and the dimer-deficient mutant (ddm); ΔCR1/I682Q/V924R) as previously described.²⁷⁾

Immunoblotting

Whole cell lysates were prepared as previously described,²⁸⁾ resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred to an Immobilon-P nylon membrane (Merk KGaA). The membrane was treated with Block Ace (KAC, Hyogo, Japan) and incubated with the primary antibodies described above. Antibodies were detected using horseradish peroxidase-conjugated anti-rabbit or mouse immunoglobulin G (Dako, Agilent Technologies, Santa Clara, CA, U.S.A.) and visualized with an enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Some antibody reactions were performed in Can Get Signal solution (Toyobo, Tokyo, Japan).

RNA Interference

Small interfering RNAs (siRNAs) against CTLC encoding CHC (HSS102017) and Silencer™ Negative Control No. 1 siRNA (4404021) were purchased from Thermo Fisher Scientific. HeLa cells were transfected with siRNAs at a final concentration of 50 nM using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were used in experiments 72 h post-transfection.

Immunofluorescence

Cells were seeded on glass coverslips (Matsunami Glass, Osaka, Japan). Two days after seeding, cells were incubated with inhibitors and ligands or were transfected with siRNAs. Cells were rinsed in cold phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde at room temperature for 15 min. After fixation, cells were permeabilized in PBS containing 0.5% Triton X-100 and washed by PBS for intracellular EGFR staining. This step was omitted for cell surface staining. Clones LA1 and A-10 were used in the immunofluorescence staining of EGFR on the cell surface and in the cytoplasm, respectively. Cells were then incubated for 50 min with primary antibodies and incubated with isotype-specific secondary antibodies conjugated with Alexa Fluor dyes (Thermo Fisher Scientific) for 30 min. These antibodies were diluted in PBS containing 0.5% bovine serum albumin (BSA). Microscopy was performed using a Zeiss LSM 700 confocal microscope (Oberkochen, Germany).

RESULTS

Negative Feedback Inhibition of EGFR

According to previous studies,²⁶⁾ we used A431 cells to examine the effects of 9,10-PQ on the activation of EGFR. Since this cell line is known to overexpress EGFR, the typical auto-phosphorylation site, Tyr-1068 was constitutively phosphorylated even in the absence of a ligand. The treatment with 9,10-PQ activated ERK within 5 min; however, the phosphorylation of Tyr-1068 did not appear to be increased. In contrast, the phosphorylation of EGFR at Thr-669, an ERK-targeted negative feedback site^14,20) gradually increased (Fig. 1A). Therefore, we examined the effects of 9,10-PQ on the inactivation of EGFR in the EGFR-overexpressing breast cancer cell line MDA-MB-468. The 9,10-PQ-induced phosphorylation of ERK and Thr-669 was accompanied by the dephosphorylation of Tyr-1068, thereby confirming the negative feedback regulation of EGFR (Fig. 1B). In addition, these negative feedback reactions occurred in a concentration-dependent manner (Fig. 1C), and were restored by the mitogen-activated protein extracellular kinase (MEK) inhibitor trametinib (Fig. 1D). Collectively, these results indicated that 9,10-PQ preferentially suppressed constitutively phosphorylated EGFR via the MEK-ERK pathway in typical EGFR-overexpressing cancer cells.

Fig. 1. Feedback Regulation of EGFR by 9.10-PQ

(A) A431 cells were grown until 80–100% confluence and then starved in serum-free media for 24 h. Cells were exposed to 9,10-PQ at 10 µM for the indicated periods. (B, C) Growing MDA-MB-468 cells were treated with 9,10-PQ at the indicated concentrations and periods. (D) MDA-MB-468 cells were pretreated with trametinib (TM; 0.03 µM) for 30 min, and then stimulated with 10 µM 9,10-PQ. (A–D) Whole cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Tyr-1068), total EGFR, phospho-ERK, and tubulin antibodies.

p38-Mediated Phosphorylation of EGFR

We previously reported that p38 induced the endocytosis of EGFR by phosphorylating serine/threonine residues, including Ser-1015, around the clathrin-recognition LL motif.^11,15) Therefore, we investigated whether the endocytosis of EGFR occurred even with the 9,10-PQ treatment in HeLa and A549 cells, which normally, but not highly, express EGFR. 9,10-PQ induced the activation of p38 as well as the phosphorylation of Ser-1015 in a concentration-dependent manner (Fig. 2A). In contrast, the phosphorylation of Tyr-1068 was not affected in HeLa cells (Fig. 2A). Similar results were obtained in A549 alveolar basal epithelial adenocarcinoma cells (Fig. 2B). The non-canonical phosphorylation of EGFR was sustained for at least 60 min (Fig. 2C). Furthermore, the 9,10-PQ-induced phosphorylation of Ser-1015 was similar that of TNF-α, and both were suppressed by the p38 inhibitor SB203580, but not by the EGFR tyrosine kinase inhibitor gefitinib (Fig. 2D).

Fig. 2. Phosphorylation of EGFR at Ser-1015 by 9,10-PQ

HeLa (A) or A549 (B) cells were treated with 9,10-PQ at the indicated concentrations for 30 min or 10 ng/mL EGF for 10 min. (C) HeLa cells were treated with 10 µM 9,10-PQ for the indicated periods. (D) HeLa cells were pretreated with SB203580 (SB; 10 µM) and gefitinib (G; 1 µM) for 30 min, and then stimulated with 10 µM 9,10-PQ for 30 min or 20 ng/mL TNF-α for 10 min. Whole cell lysates were immunoblotted with phospho-EGFR (Ser-1015), total EGFR, phospho-p38, and tubulin antibodies.

CME of EGFR

The cell surface and intracellular localization of EGFR was investigated by immunofluorescence under non-permeable and permeable conditions, respectively. A stimulation with 9,10-PQ and TNF-α strongly induced the internalization of EGFR, which was detected by its disappearance on the plasma membrane and sorting to intracellular endosomes (Fig. 3A). In addition, endocytosed EGFR completely overlapped with phospho-Ser-1015 staining, both of which were inhibited by SB203580, but not by gefitinib (Fig. 3B). We also investigated the involvement of non-canonical mechanisms using EGFR-KO HeLa cells reconstituted with wild-type or mutant EGFR. Mutant R1m has amino acid substitutions in the serine/threonine residues with alanine, and, thus, lacks non-canonical mechanisms. On the other hand, ddm has deletions and substitutions in the extracellular and intracellular regions required for ligand binding and dimerization; therefore, only non-canonical mechanisms occur. Figure 3C shows that the internalization of wild-type and ddm EGFR, but not the R1m mutant, was induced by 9,10-PQ. Furthermore, the knockdown of the clathrin heavy chain impaired 9,10-PQ- and TNF-α-induced endocytosis. These results indicate that 9,10-PQ, similar to TNF-α, induced the non-canonical mechanism of the CME of EGFR.

Fig. 3. Non-canonical Endocytosis of EGFR by 9,10-PQ

(A) HeLa cells were stimulated with 10 µM 9,10-PQ for 30 min or 20 ng/mL TNF-α for 15 min. The localization of EGFR on the cell surface (upper panel) or in the cytoplasm (lower panel) was investigated by immunofluorescence. (B) HeLa cells were pretreated with 1 µM gefitinib or 10 µM SB203580 for 30 min, and then stimulated with 10 µM 9,10-PQ for 30 min or 20 ng/mL TNF-α for 15 min. The localization of pS-EGFR and EGFR in permeabilized cells was investigated by immunofluorescence. (C, D) EGFR-KO HeLa cells re-expressing wild-type and mutant EGFR (WT, R1m, and ddm) (C) and HeLa cells transfected with negative control or CHC siRNAs (D) were stimulated with 9,10-PQ and TNF-α. The localization of EGFR was investigated by immunofluorescence. Scale bar = 10 µm.

DISCUSSION

The non-canonical regulation of ligand-unbound EGFR is gradually being recognized as an important advance in EGFR biology. In EGFR-overexpressing human cancer cells, we demonstrated that 9,10-PQ induced the MEK/ERK-mediated phosphorylation of EGFR at Thr-669, resulting in the feedback inactivation of EGFR. However, this result is inconsistent with previous findings showing that 9,10-PQ activated EGFR via the ROS-dependent inhibition of PTP1B. Other atmospheric electrophiles, including 1,2-naphotoquinone (1,2-NQ) and 1,4-NQ, have also been reported to activate EGFR.^29,30) It currently remains unknown why this difference occurred; however, previous studies showed that ROS activated ERK in a PTP1B-independent manner.^31,32) Therefore, further studies are needed to investigate the expression of ligands that may influence the activation of EGFR by inhibiting PTP1B. In any case, the main difference between these completely opposite observations is whether the activation of ERK by 9,10-PQ is mediated by EGFR. If ERK is activated independently of EGFR, it is reasonable that the negative feedback inhibition found in this study occurs in preference to the activation of EGFR. Since the negative feedback mechanism has been confirmed in various cancer cells, further analyses of the intracellular and extracellular environments in which electron donors, such as 9,10-PQ, regulate the activation or inactivation of EGFR are required. The effects of 9,10-PQ on the negative feedback mechanism in melanoma cells harboring the BRAF-V600E mutation also need to be clarified because it is involved in the reactivation of ERK associated with BRAF inhibitor resistance.³³⁾

The present study also revealed the p38-mediated CME of EGFR by 9,10-PQ. Since it was not inhibited by gefitinib, this signaling pathway is completely independent of EGFR tyrosine kinase activity. The function of CME in the activation of EGFR currently remains unclear. However, changes in the CME of EGFR have important roles, such as increasing the intensity of signal transduction pathways; therefore, many studies have been conducted with the aim of elucidating the mechanisms underlying CME. For example, clathrin-mediated EGFR endocytosis was shown to play a role in gefitinib-refractory wild-type EGFR NSCLC cell lines.³⁴⁾ Furthermore, metastasis-associated in colon cancer 1 (MACC1) was found to regulate CME and subsequent recycling to the plasma membrane, inducing EGFR-mediated signaling and increased cell proliferation.³⁵⁾ Moreover, CME was demonstrated in mouse tumor xenograft models in vivo. It has not yet been established whether CME is caused by the non-canonical mechanism mediated by p38; however, it may be involved in malignant transformation and cancer progression induced by 9,10-PQ. We previously reported the anti-apoptotic role of p38-mediated EGFR CME, which further supports the notion that 9,10-PQ utilizes the non-canonical EGFR pathway.¹⁶⁾

A positive relationship has been demonstrated between air pollutants and the onset and progression of various cancers, such as bladder cancer and lung cancer.^36,37) Air pollution is still considered to be a major cause of human cancer, for which EGFR is an important target; therefore, further studies are warranted on their functional relationships in the tumor microenvironment.

Acknowledgments

This work was supported in part by JSPS KAKENHI Grant Nos. 19H03368 and 22H02763, JST SPRING Grant No. JPMJSP2145 and research Grants from Takeda Science Foundation.

Author Contributions

NY and HS conceived and designed the experiments. NY and JT performed the experiments. YZ and SY analyzed the data. TM contributed materials. NT and HS wrote the manuscript. YZ, SY, and TM critically reviewed the manuscript. All authors had final approval of the submitted version.

Conflict of Interest

The authors declare no conflict of interest.

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